EP2357188A1 - Linker and support for solid phase synthesis of nucleic acid - Google Patents

Linker and support for solid phase synthesis of nucleic acid Download PDF

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Publication number
EP2357188A1
EP2357188A1 EP10251831A EP10251831A EP2357188A1 EP 2357188 A1 EP2357188 A1 EP 2357188A1 EP 10251831 A EP10251831 A EP 10251831A EP 10251831 A EP10251831 A EP 10251831A EP 2357188 A1 EP2357188 A1 EP 2357188A1
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EP
European Patent Office
Prior art keywords
nucleic acid
group
solid phase
support
synthesis
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German (de)
English (en)
French (fr)
Inventor
Yoshihiro Hayakawa
Masaki Tsukamoto
Kenjiro Mori
Kenjiro Minomi
Eri Maeta
Tatsuya Konishi
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Nagoya University NUC
Nitto Denko Corp
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Nagoya University NUC
Nitto Denko Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/34—Esters of acyclic saturated polycarboxylic acids having an esterified carboxyl group bound to an acyclic carbon atom
    • C07C69/40—Succinic acid esters
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B80/00—Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a linker to be used for solid phase synthesis of nucleic acid, a support for solid phase synthesis carrying the linker, and a production method of nucleic acid using the support.
  • nucleic acid is generally synthesized by the following steps.
  • nucleoside to be the 3' terminal of the nucleic acid to be synthesized is ester bonded to a cleaving linker such as succinyl group and the like via 3'-OH group so that the nucleoside is previously carried on a support for solid phase synthesis (nucleoside linker).
  • a synthesis reaction comprising the following steps is generally performed in the reaction column according to a synthesis program of the automated nucleic acid synthesizer:
  • nucleoside When the above-mentioned synthesis is performed, as mentioned above, it is necessary to carry, in advance, nucleoside to be the 3' terminal (starting material) on a support for solid phase synthesis via a cleaving linker. Moreover, the 3' terminal varies depending on the sequence of nucleic acid desired to be synthesized. In the case of DNA oligonucleotide, 4 kinds of dA, dG, dC, dT are necessary, and in the case of RNA, 4 kinds of rA, rG, rC, rU are also necessary. For synthesis of modified oligonucleotide, a support for solid phase synthesis previously carrying a modified nucleoside is necessary, making the process complicated.
  • a support for solid phase synthesis carrying a universal linker (universal support) has been developed as a linker to connect a solid phase support and a starting material, in the place of nucleoside ⁇ succinyl linker and the like generally used heretofore.
  • the process includes, irrespective of the kind of nucleoside or nucleotide for the 3' terminal of nucleic acid desired to be synthesized, reacting nucleoside phosphoramidite to be the 3' terminal in the same step as general automated nucleic acid synthesis to start the synthesis and, after synthesizing the desired nucleic acid, cleaving the nucleic acid from the support for solid phase synthesis by a method similar to a general method. It is not necessary to prepare a support for solid phase synthesis carrying various nucleoside-linkers as mentioned above.
  • the protecting group of these nucleophilic groups are also dissociated to attack the 3' terminal phosphorus, and the phosphate group is cleaved from the 3' terminal to form cyclophosphoric acid. All are used to synthesize nucleic acid having an -OH group at the 3' terminal.
  • some universal supports have been proposed for providing a phosphate group at the 5' terminal or 3' terminal of nucleic acid desired to be synthesized.
  • patent document 5 and non-patent document 4 disclose a universal linker capable of synthesizing nucleic acid having a phosphate group at the 5'terminal, a universal support carrying the universal linker, and a synthesis method of nucleic acid using the universal support. Since a nucleic acid having a phosphate group at the 5' terminal or 3' terminal can be widely used in various fields of biochemistry such as chemical linkage reaction of nucleic acid, modification of nucleic acid utilizing terminal phosphate group, structural study of nucleic acid and the like, it is extremely useful. In view of such situation, a universal linker capable of synthesizing a nucleic acid having a phosphate group at the 5' terminal or 3' terminal, and a universal support carrying the linker are desired.
  • the present invention aims to provide a universal linker capable of synthesizing a nucleic acid having a phosphate group at the 3' terminal, a universal support carrying the linker, and a method for synthesizing a nucleic acid using the universal support.
  • the present invention aims to provide a support for solid phase synthesis, which is capable of universally synthesizing a nucleic acid having a phosphate group at the 3' terminal in a high purity, which does not require a support for solid phase synthesis with modified nucleoside carried in advance even when synthesizing a modified oligonucleotide.
  • a linker for solid phase synthesis of nucleic acid comprising a compound represented by at least one of the following formulae
  • X is a hydroxyl-protecting group dissociated by acid
  • L is a linkage moiety cleaved by alkali
  • R 1 , R 2 , R 3 and R 4 are each independently a hydrogen atom, an alkyl group having a carbon number of 1 - 6, an alkoxy group having a carbon number of 1 - 6, an alkylamino group having a carbon number of 1 - 6 or a halogen atom.
  • X is a hydroxyl-protecting group dissociated by acid
  • L is a linkage moiety cleaved by alkali
  • Sp is a solid phase support
  • R 1 , R 2 , R 3 and R 4 are each independently a hydrogen atom, an alkyl group having a carbon number of 1 - 6, an alkoxy group having a carbon number of 1 - 6, an alkylamino group having a carbon number of 1 - 6 or a halogen atom.
  • [6] The support for solid phase synthesis of nucleic acid according to the above-mentioned [4] or [5], wherein X is a dimethoxytrityl group.
  • a method of producing a nucleic acid comprising a step of performing a nucleic acid synthesis reaction on the support for solid phase synthesis of nucleic acid according to any of the above-mentioned [4] to [7].
  • [9] The production method of the above-mentioned [8], wherein the nucleic acid synthesis reaction is performed by a solid phase phosphoramidite method.
  • the linker for solid phase synthesis of nucleic acid of the present invention is a universal linker, and the support for nucleic acid synthesis of the present invention, carrying the linker is a universal support.
  • the support for nucleic acid synthesis carrying the linker of the present invention does not require a support for solid phase synthesis carrying a nucleotide to be the 3' terminal of the nucleic acid desired to be synthesized, and can synthesize a nucleic acid having a phosphate group introduced into the 3' terminal at a high purity.
  • the support for nucleic acid synthesis of the present invention is preferably used for automatic synthesis of a nucleic acid having a phosphate group at the 3' terminal. Furthermore, the production method of a nucleic acid using the support for nucleic acid synthesis of the present invention does not require separate introduction of a phosphate group into the 3' terminal of a synthesized nucleic acid, unlike conventional methods.
  • nucleic acid refers to a linear compound (oligonucleotide) wherein nucleotides are connected via phosphodiester bonds, and is understood to encompass DNA, RNA and the like.
  • the nucleic acid may be single-stranded or double-stranded. Since it allows an efficient synthesis using a nucleic acid synthesizer, the nucleic acid is preferably single-stranded.
  • nucleic acid in the present specification includes not only a oligonucleotide containing a purine base such as adenine (A), guanine (G) and the like and a pyrimidine base such as thymine (T), cytosine(C), uracil (U) and the like but also a modified oligonucleotide containing other modified heterocyclic base.
  • a purine base such as adenine (A), guanine (G) and the like
  • a pyrimidine base such as thymine (T), cytosine(C), uracil (U) and the like
  • T thymine
  • C cytosine
  • U uracil
  • the nucleotide length of the nucleic acid is not particularly limited, and the nucleic acid is preferably 2 to 200 nucleotides long. If the nucleic acid is too long, the yield and purity of the nucleic acid obtained decrease.
  • linker in the present specification refers to a molecule that links two substances via a covalent bond.
  • the linker connects a solid phase support and a nucleic acid.
  • the linker for solid phase synthesis of nucleic acid of the present invention is represented by the following formula.
  • L is a linkage moiety cleaved by alkali, and examples thereof include, but are not limited to, a succinyl group (succinyl linker), Q linker ( Pon et al., Nucleic Acids Res., 27, 1531 (1999 )) and the like.
  • a succinyl linker is preferably used. These linkage moieties can be easily cleaved by hydrolysis with aqueous ammonia, aqueous ammonia/methylamine mixture and the like, and oligonucleotide is cleaved from a support for solid phase synthesis after completion of automatic synthesis.
  • R 1 , R 2 , R 3 and R 4 are each independently a hydrogen atom, an alkyl group having a carbon number of 1 - 6, an alkoxy group having a carbon number of 1 - 6, an alkylamino group having a carbon number of 1 - 6 or a halogen atom.
  • Examples of the alkyl group having a carbon number of 1 - 6 include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-amyl group, an isoamyl group, a sec-amyl group, a tert-amyl group, a hexyl group and the like.
  • a methyl group or an ethyl group is preferable.
  • Examples of the alkoxy group having a carbon number of 1 - 6 include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group, a hexyloxy group and the like. Among these, a methoxy group is preferable.
  • Examples of the alkylamino group having a carbon number of 1 - 6 include a methylamino group, a dimethylamino group, an ethylamino group, a diethylamino group, a propylamino group, a dipropylamino group, an isopropylamino group, a diisopropylamino group and the like. Among these, a dimethylamino group is preferable.
  • examples of the halogen atom include chlorine, fluorine, bromine, iodine and the like. Among these, chlorine or fluorine is preferable.
  • the solid phase support for solid phase synthesis of nucleic acid of the present invention carries the above-mentioned linker for solid phase synthesis of nucleic acid of the present invention, and is represented by the following formula
  • Sp is a solid phase support.
  • the solid phase support is not particularly limited as long as a reagent used in excess can be easily removed by washing and, for example, glass porous support, porous synthetic polymer support such as polystyrene support, acrylamide support etc., and the like can be mentioned.
  • Sp is a glass porous support.
  • the "glass porous support” refers to a porous support containing glass as a constituent component and examples thereof include, but are not limited to, porous glass particles in a granular shape (CPG) and the like. More specifically, as the aforementioned CPG, a CPG solid phase support having a long chain aminoalkyl spacer (LCAA-CPG solid phase support) is preferably used, and further, for the synthesis of a long chain nucleotide, one having a CPG pore of preferably 20 - 400 nm, more preferably 50 - 200 nm, most preferably 100 nm, is used.
  • CPG CPG solid phase support having a long chain aminoalkyl spacer
  • Sp is a polystyrene support.
  • polystyrene support is a solid phase support constituted with a copolymer containing structural unit (A) represented by the following formula:
  • substituted derivatives of the structural unit (A) include a compound wherein one or more hydrogen atoms contained in the structural unit (A) (including hydrogen atom of benzene ring) are substituted by an alkyl group having a carbon number of 1 - 6 (e.g., methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-amyl group, isoamyl group, sec-amyl group, tert-amyl group), a halogen atom, an amino group, a hydroxy group, a carboxyl group, a sulfone group, a cyano group, a methoxy group, a nitro group, a vinyl group and the like.
  • an alkyl group having a carbon number of 1 - 6 e.g., methyl group, ethyl group,
  • the substituent is preferably an amino group or a hydroxy group.
  • the position of the substituent is not particularly limited, and is preferably the para-position with respect to the main chain on the benzene ring.
  • a preferable substitution derivative of the structural unit (A) is the hydroxystyrene structural unit (B) shown in the following formula.
  • the amount of structural unit (A) and/or substitution derivatives thereof relative to the total amount of the structural units contained in the copolymer constituting the polystyrene support is not particularly limited, and is generally 50 to 100% by weight, preferably 60 to 100% by weight.
  • the monomer compound copolymerizable with the above-mentioned structural unit (A) include, but are not limited to, styrene; nucleus alkyl substituted styrene such as o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, ethylstyrene, trimethylstyrene, and p-t-butylstyrene and the like; ⁇ -alkyl substituted styrene such as ⁇ -methylstyrene, ⁇ -methyl-p-methylstyrene and the like; nucleus halogenated styrene such as chlorostyrene, dichlorostyrene, fluorostyrene, pentafluorostyrene, bromostyrene and the like; alkyl halide styrene such as chloromethylstyrene
  • alkyl (meth)acrylate monomer such as methyl(meth)acrylate, ethyl(meth)acrylate, propyl(meth)acrylate, butyl(meth)acrylate and the like
  • vinyl cyanide monomer such as acrylonitrile, methacrylonitrile, ethacrylonitrile etc., and the like.
  • Sp may be a solid phase support consisting of styrene-hydroxystyrene-divinylbenzene copolymer particles ( JP-A-2005-097545 , JP-A-2005-325272 and JP-A-2006-342245 ) or a styrene-(meth)acrylonitrile-hydroxystyrene-divinylbenzene copolymer ( JP-A-2008-074979 ), having, in addition to the above-mentioned structural units (A) and (B), divinylbenzene structural unit (C) represented by the following formula:
  • one or more hydrogen atoms (including hydrogen atom of benzene ring) in the above-mentioned divinylbenzene structural unit (C) may be substituted by an alkyl group having a carbon number of 1 - 6 (e.g., methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-amyl group, isoamyl group, sec-amyl group, tert-amyl group), a halogen atom, an amino group, a hydroxy group, a carboxyl group, a sulfone group, a cyano group, a methoxy group, a nitro group, a vinyl group and the like.
  • the substituent is preferably an amino group or a hydroxy group.
  • the structural unit of (meth)acrylonitrile may contain each or both of the structural units of acrylonitrile and methacrylonitrile.
  • the amount of the structural unit of (meth)acrylonitrile to the total amount of the structural unit of the styrene-(meth)acrylonitrile-hydroxystyrene-divinylbenzene copolymer is too high or too low, the degree of swelling greatly varies depending on the kind of organic solvent. Therefore, the amount is preferably 2 - 11 mmol/g.
  • Sp is an acrylamide support. More specifically, in the above-mentioned formula, Sp may be a support for solid phase synthesis, which is comprised of, in addition to the above-mentioned structural units (A) and (C), an aromatic monovinyl compound-divinyl compound-(meth)acrylamide derivative copolymer further containing a (meth)acrylamide derivative monomer.
  • the aforementioned aromatic monovinyl compound is not particularly limited.
  • styrene nucleus alkyl substituted styrene such as o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, ethylstyrene, trimethylstyrene, p-t-butylstyrene and the like; ⁇ -alkyl substituted styrene such as ⁇ -methylstyrene, ⁇ -methyl-p-methylstyrene and the like; nucleus halogenated styrene such as chlorostyrene, dichlorostyrene, fluorostyrene, pentafluorostyrene, bromostyrene and the like; alkyl halide styrene such as chloromethylstyrene, fluoromethylstyrene and the like; hydroxystyrene; hydroxymethylstyrene; vinyl
  • Examples of the aforementioned (meth)acrylamide derivative include, but are not limited to, N-alkyl(meth)acrylamide; N,N-dialkyl(meth)acrylamide; N-alkoxyalkyl(meth)acrylamide; 2-(meth)acrylamide alkane sulfonic acid; N-alkylol(meth)acrylamide such as N-methylol(meth)acrylamide and the like; acrylamide; methacrylamide; diacetone acrylamide; N,N-dimethyl aminopropylacrylamide; acryloylmorpholine; N-phenoxymethyl(meth)acrylamide and the like.
  • the alkyl contained in the aforementioned N-alkyl(meth)acrylamide is generally a linear or branched alkyl having a carbon number of 1 - 6, preferably 1 - 3.
  • the N-alkyl(meth)acrylamide include N-methyl(meth)acrylamide, N-ethyl(meth)acrylamide, N-n-propyl(meth)acrylamide, N-isopropyl(meth)acrylamide, N-tert-butyl(meth)acrylamide, and N-lauryl (meth)acrylamide and the like.
  • the two alkyls contained in the aforementioned N,N-dialkyl(meth)acrylamide are each generally a linear or branched alkyl having a carbon number 1 - 6, preferably 1 - 3.
  • Examples of the N,N-dialkyl(meth)acrylamide include N,N-dimethyl(meth)acrylamide, N,N-diethyl(meth)acrylamide, N,N-diisopropyl(meth)acrylamide, N,N-di-tert-butyl(meth)acrylamide, N,N-dilauryl (meth)acrylamide, N,N-di-tert-octyl(meth)acrylamide, N,N-dilauryl (meth)acrylamide, N,N-dicyclohexyl(meth)acrylamide and the like.
  • the alkoxy contained in the aforementioned N-alkoxyalkyl(meth)acrylamide is generally a linear or branched alkoxy having a carbon number of 1 - 6, preferably 1 - 3.
  • the alkyl contained in N-alkoxyalkyl(meth)acrylamide is generally a linear or branched alkyl having a carbon number of 1 - 6, preferably 1 - 3.
  • N-alkoxyalkyl(meth)acrylamide examples include N-methoxymethyl(meth)acrylamide, N-ethoxymethyl(meth)acrylamide, N-propoxymethyl(meth)acrylamide, N-butoxy methyl(meth)acrylamide, N-ethoxyethyl(meth)acrylamide, N-methoxypropyl(meth)acrylamide, N-ethoxypropyl(meth)acrylamide, N-isopropoxyethyl(meth)acrylamide and the like.
  • the alkane contained in the aforementioned 2-(meth)acrylamide alkanesulfonic acid is generally a linear or branched alkane having a carbon number of 1 - 6, preferably 1 - 3.
  • Examples of the 2-(meth)acrylamide alkanesulfonic acid include 2-acrylamide propanesulfonic acid, 2-acrylamide-n-butanesulfonic acid, 2-acrylamide-n-hexanesulfonic acid, 2-acrylamide-2-methylpropanesulfonic acid and the like.
  • the aforementioned (meth)acrylamide derivative is preferably diacetone acrylamide, N-isopropyl(meth)acrylamide, N-methoxymethyl(meth)acrylamide or N,N,-dimethyl(meth)acrylamide.
  • a typical structural unit derived from (meth)acrylamide derivative monomer includes the following structural units.
  • the porous particle to be used as the support for solid phase synthesis of the present invention is preferably one having a functional group that contributes to nucleic acid synthesis.
  • the "contributes to nucleic acid synthesis” refers to a functional group which can be the starting point of nucleic acid synthesis and to which a linker can be added. Specifically, an amino group, a hydroxy group and the like can be mentioned.
  • the content of the functional group that contributes to nucleic acid synthesis is not particularly limited. When the content of the functional group is too small, the yield of nucleic acid decreases and when it is too high, the purity of the obtained nucleic acid decreases. Therefore, it is preferably 10 - 2000 ⁇ mol/g, more preferably 50 - 1000 ⁇ mol/g, further preferably 100 - 800 ⁇ mol/g.
  • the amount of the hydroxyl group of the porous particles of the present invention is measured by titration based on JIS K0070.
  • an acetylation reagent is produced by adding pyridine to 25 g of acetic anhydride to the total amount of 100 mL.
  • the above-mentioned acetylation reagent (0.5 mL), pyridine (4.5 mL) and a sample (about 0.5 g) are placed in a flask, and the mixture is heated at 95 - 100°C for 2 hr to acetylate the hydroxyl group.
  • distilled water (1 mL) is added into the flask, and the mixture is heated to decompose acetic anhydride that was not consumed by acetylation to acetic acid.
  • the amount of the acetic acid is measured by neutralization titration using 0.5 mol/L of aqueous potassium hydroxide solution. Separately, a blank measurement is performed in the same way, but without adding the sample.
  • the amount of the linker to be bound to the solid phase support is not particularly limited.
  • the linker content is too low, the yield of nucleic acid decreases.
  • the linker content is too high, the purity of the nucleic acid obtained tends to decrease and the number of nucleotides of the obtained nucleic acid tends to decrease. Therefore, it is preferably within the range of 20 - 800 ⁇ mol/g, more preferably 25 - 500 ⁇ mol/g.
  • the shape of the aforementioned porous solid phase support is not particularly limited and may be any shape of plate, particle, fiber and the like. Since the packing efficiency to a synthesis reaction container can be enhanced, and the reaction container is not easily broken, a porous synthetic polymer having a particle shape is preferable.
  • the term "particle” in the specification does not mean being exactly spherical, but means having any constant form (e.g., roughly spherical forms such as ellipse spherical, polygonal form, cylindrical form, irregular forms such as konpeito form, and the like).
  • the size (volume) of one particle of the porous synthetic polymer particles is not particularly limited, when the average particle size measured by laser diffraction (scattering type) of the porous particles is smaller than 1 ⁇ m, inconvenience occurs when it is packed in a column and used in that the back pressure becomes too high or a solution sending rate becomes slow.
  • the average particle size is more than 1000 ⁇ m, the gap between the support particles becomes large and efficient packing of support particles in a column having a predetermined volume becomes difficult. Therefore, it is preferably 1 - 1000 ⁇ m, more preferably 5 - 500 ⁇ m, further preferably 10 - 200 ⁇ m.
  • the specific surface area of the aforementioned synthetic polymer particles measured by a multi-point BET method is not particularly limited, when the specific surface area is less than 0.1 m 2 /g, the degree of swelling in an organic solvent becomes low, and a synthesis reaction tends to be difficult to occur. On the other hand, when it is more than 500 m 2 /g, fine pore size becomes small, and a synthesis reaction tends to be difficult to occur. Therefore, the specific surface area is preferably 0.1 - 500 m 2 /g, more preferably 10 - 300 m 2 /g, further preferably 50 - 200 m 2 /g.
  • the average fine pore size of the aforementioned synthetic polymer particles measured by a mercury intrusion technique is not particularly limited.
  • the pore size is too small, the field of the synthesis reaction becomes small and a desired reaction does not occur easily, or the nucleotide length tends to be less than a desired number.
  • the pore size is too large, the frequency of contact between a hydroxyl group and a substance involved in the reaction on the surface of polymer particles, which is the reaction field, decreases to lower the yield. Therefore the average fine pore size is preferably 1 - 200 nm, more preferably 5 - 100 nm, more preferably 20 - 70 nm.
  • Sp is a low swelling cross-linking polystyrene particle commercially available as NittoPhase (registered trade mark) (manufactured by NITTO DENKO Co., Ltd.).
  • NittoPhase registered trade mark
  • a solid phase nucleic acid synthesis method using NittoPhase is preferably used since it shows a small peak area due to impurity and guarantees high yield and high purity in a wide scale from labo scale to mass synthesis system.
  • the production method of the support for solid phase synthesis of nucleic acid of the present invention is not particularly limited, for example, the support can be produced by the following method.
  • An aromatic ring compound having a hydroxy group and a hydroxymethyl group at the ortho-position or para-position e.g., 2-hydroxybenzyl alcohol, 4-hydroxybenzyl alcohol
  • 4-hydroxybenzyl alcohol is reacted with 4,4'-dimethoxytrityl chloride and the like to protect the hydroxymethyl group.
  • this compound is reacted with succinic anhydride together with triethylamine to bind a succinyl linker moiety to a hydroxy group, which is then bound with a solid phase support having -OH group or -NH 2 group to give the support for solid phase synthesis of nucleic acid of the present invention.
  • nucleic acid automatic synthesizer For nucleic acid synthesis using the support for solid phase synthesis of nucleic acid of the present invention, a nucleic acid automatic synthesizer is used and various synthesis methods known per se can be used.
  • the "nucleic acid synthesis reaction” particularly means an elongation reaction of nucleotide constituting a nucleic acid.
  • nucleotides are sequentially bound to a nucleoside, nucleotide or oligonucleotide bound to the solid phase support, whereby an elongated oligonucleotide is obtained.
  • the nucleic acid synthesis reaction can be performed by the H-phosphonate method, phosphoester method, solid phase phosphoramidite method and the like. Of these, since high capacity of synthesizing nucleic acid and high purity of nucleic acid obtained, a solid phase phosphoramidite method is preferable.
  • a preferable embodiment of the nucleic acid synthesis reaction by a solid phase phosphoramidite method includes, for example, a method consisting of each of the following steps:
  • a chemical reaction shown by the following formula occurs, and a nucleic acid having a phosphate group at the 3' terminal is produced.
  • the chemical reaction shown by the following formula occurs, and a nucleic acid having a phosphate group at the 3' terminal is produced.
  • nucleic acid synthesis method using support (B) for solid phase synthesis of nucleic acid of the present invention which is shown in Example 4 as a preferable embodiment of the present invention, a chemical reaction shown by the following formula occurs, and a nucleic acid having a phosphate group at the 3' terminal is produced.
  • Examples of the activating agent to be used in the nucleic acid production method of the present invention include, but are not limited to, 1H-tetrazole, 4,5-dicyanoimidazole, 5-ethylthio-1H-tetrazole, benzimidazolium triflate (BIT), N-phenylbenzimidazoliumtriflate, imidazoliumtriflate (IMT), N-PhIMP, 5-nitrobenzimidazoliumtriflate, triazoliumtriflate, 1-hydroxybenzotriazole (HOBT), N-(cyanomethyl)pyrrolidinium tetrafluoroborate and the like.
  • BIT benzimidazolium triflate
  • IMT imidazoliumtriflate
  • N-PhIMP N-nitrobenzimidazoliumtriflate
  • HOBT 1-hydroxybenzotriazole
  • N-(cyanomethyl)pyrrolidinium tetrafluoroborate and the like.
  • a porous polystyrene solid phase support having a hydroxyl group (NittoPhase, manufactured by NITTO DENKO Co., Ltd. (registered trade mark)) was dispersed in acetonitrile, and the aforementioned compound, HBTU and N,N-diisopropylethylamine were added. The mixture was reacted at 28°C for 23 hr to carry the aforementioned compound on the solid phase support.
  • the binding amount of the linker of the present invention to the solid phase support obtained as mentioned above was 32 ⁇ mol/g.
  • the solid phase support (A) for nucleic acid synthesis of the present invention (31 mg) prepared in Example 1 was packed in a reaction column, 5-mer thymidine (5'-TTTTT-3') was synthesized by DMT-off (method of removing 5' terminal-protecting group) (synthesis scale 1 ⁇ mol) using an automated DNA/RNA synthesizer ABI3400 (manufactured by Applied Biosystems). After the synthesis, the solid phase support bound with DNA oligonucleotide was immersed in a 30% aqueous ammonia/ethanol (2:1) mixed solution at 55°C for 14 hr to cleave the DNA oligonucleotide from the solid phase support.
  • 20-mer DNA oligonucleotide (5'-ATA CCG ATT AAG CGA AGT TT-3':SEQ ID NO: 1) was synthesized by DMT-off (synthesis scale 1 ⁇ mol) using solid phase support (A) for nucleic acid synthesis of the present invention. After synthesis, the DNA oligonucleotide was cleaved from the solid phase support.
  • cleavable linker DMT-dT-3'-succinate manufactured by Beijing OM Chemicals
  • a commercially available solid phase support NittoPhase registered trade mark
  • the binding amount of the compound to the solid phase support was 38 ⁇ mol/g.
  • the solid phase support (27 mg) was packed in a reaction column, 5-mer DNA oligonucleotide (5'-TTTTT-3') was synthesized by DMT-off in the same manner as in Example 1 (synthesis scale 1 ⁇ mol). After synthesis, the DNA oligonucleotide was cleaved from the solid phase support.
  • 20-mer DNA oligonucleotide (5'-ATA CCG ATT AAG CGA AGT TT-3':SEQ ID NO: 1) was synthesized by DMT-off (synthesis scale 1 ⁇ mol) using the solid phase synthesis support bound with a cleaving linker DMT-dT-3'-succinate, which was produced in Comparative Example 1. After synthesis, the DNA oligonucleotide was cleaved from the solid phase support.
  • the DNA oligonucleotide solutions obtained in Examples 2 and 3, as well as Comparative Examples 1 and 2 were measured by high performance liquid chromatography (HPLC) (measurement conditions: column; Clarity 3 ⁇ m Oligo-RP 50 ⁇ 4.6 mm (manufactured by Phenomenex), UV detection; 260 nm, Buffer A; 100 mM TEAA (pH 7.0), Buffer B; acetonitrile).
  • Fig. 1A - D show each HPLC chart.
  • the main peak of the nucleic acid produced in Example 3 was confirmed to be 20-mer oligonucleotide having a phosphate group at the 3' terminal (molecular weight (measured value); 6219).
  • the main peaks of the DNA oligonucleotides produced in Comparative Examples 1 and 2 were confirmed to be T-5-mers having 3' terminal-OH group (molecular weight (measured value); 1459) and 20-mer DNA oligonucleotide having 3' terminal-OH group (molecular weight (measured value); 6139), respectively.
  • NittoPhase registered trade mark (manufactured by NITTO DENKO Co., Ltd.), which is a cross-linking polystyrene solid phase support having a hydroxy group was dispersed in acetonitrile, the aforementioned compound, HBTU and N,N-diisopropylethylamine were added and the mixture was reacted at 28°C for 23 hr to carry the aforementioned compound on the solid phase support.
  • the binding amount of the linker of the present invention to the solid phase support obtained as mentioned above was 29 ⁇ mol/g.
  • the solid phase support (B) for nucleic acid synthesis of the present invention (34 mg) prepared in Example 4 was packed in a reaction column, DNA oligonucleotide 5-mer thymidine (5'-TTTTT-3') was synthesized by DMT-on (method of not removing 5' terminal-protecting group) (synthesis scale 1 ⁇ mol) using an automated DNA/RNA synthesizer ABI3400 (manufactured by Applied Biosystems). After the synthesis, the solid phase support bound with DNA oligonucleotide was immersed in a 30% aqueous ammonia/ethanol (1:1) mixed solution at 55°C for 15 hr to cleave the DNA oligonucleotide from the solid phase support.
  • 20-mer DNA oligonucleotide (5'-ATA CCG ATT AAG CGA AGT TT-3':SEQ ID NO: 1) was synthesized by DMT-on (synthesis scale 1 ⁇ mol) using solid phase support (B) for nucleic acid synthesis of the present invention. After synthesis, the DNA oligonucleotide was cleaved from the solid phase support.
  • a solid phase support (25 mg) bound with DMT-dT-3'-succinate produced in Comparative Example 1 was packed in a reaction column and, in the same manner as in Example 5, DNA oligonucleotide 5-mer thymidine (5'-TTTTT-3') was synthesized by DMT-on (synthesis scale 1 ⁇ mol). After synthesis, the DNA oligonucleotide was cleaved from the solid phase support.
  • a solid phase support (25 mg) bound with DMT-dT-3'-succinate produced in Comparative Example 1 was packed in a reaction column and, in the same manner as in Example 6, 20-mer DNA oligonucleotide (5'-ATA CCG ATT AAG CGA AGT TT-3':SEQ ID NO: 1) was synthesized by DMT-on (synthesis scale 1 ⁇ mol). After synthesis, the DNA oligonucleotide was cleaved from the solid phase support.
  • the DNA oligonucleotide solutions obtained in Examples 5 and 6, as well as Comparative Examples 3 and 4 were measured by HPLC.
  • the measurement conditions were column; XBridge OST C18 2.5 ⁇ m 50 ⁇ 4.6 mm (manufactured by Waters), UV detection; 260 nm, Buffer A; 100 mM HFIP/7 mM TEA/water (pH 8.0), Buffer B; methanol.
  • Fig. 2A - D show each HPLC chart.
  • the main peak of the DNA oligonucleotide produced in Example 6 was confirmed to be 20-mer oligonucleotide having a phosphate group at the 3' terminal (molecular weight (measured value); 6521).
  • the main peaks of the DNA oligonucleotides produced in Comparative Examples 3 and 4 were confirmed to be T-5-mers having 3' terminal-OH group (molecular weight (measured value); 1760) and 20-mer DNA oligonucleotide having 3' terminal-OH group (molecular weight (measured value); 6441), respectively.
  • Table 1 collectively shows the data obtained in Experimental Examples 1 and 2.
  • a nucleic acid having a phosphate group at the 3' terminal which is automatically synthesized using the solid phase support for nucleic acid synthesis of the present invention can be widely used for chemical linkage reaction of nucleic acid, modification of nucleic acid utilizing 3' phosphate group, structural study of nucleic acid and the like.

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EP3106519A1 (en) * 2015-06-18 2016-12-21 Nitto Denko Corporation Method for cutting out rna oligonucleotide

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JP6028979B2 (ja) * 2012-01-30 2016-11-24 日東電工株式会社 核酸固相合成用リンカー及び担体
JP7033402B2 (ja) 2017-07-05 2022-03-10 日東電工株式会社 固相核酸合成方法、固相核酸合成用溶液組成物
CN107383246A (zh) * 2017-08-28 2017-11-24 盐城师范学院 一种聚苯乙烯基质核酸固相有机合成载体及其制备方法
EP3950126A4 (en) 2019-03-29 2023-11-01 Sumitomo Chemical Company Limited INORGANIC POROUS SUPPORT AND METHOD FOR PRODUCING NUCLEIC ACIDS THEREFROM
WO2020202951A1 (ja) * 2019-03-29 2020-10-08 住友化学株式会社 無機多孔質担体、及びこれを用いた核酸の製造方法
WO2020202953A1 (ja) * 2019-03-29 2020-10-08 住友化学株式会社 無機多孔質担体、及びこれを用いた核酸の製造方法
WO2020202952A1 (ja) * 2019-03-29 2020-10-08 住友化学株式会社 無機多孔質担体、及びこれを用いた核酸の製造方法
CN110511973B (zh) * 2019-07-16 2021-05-07 杭州原合生物科技有限公司 一种用于核酸原位合成的固相载体及其制备方法
WO2023069599A1 (en) 2021-10-22 2023-04-27 Hongene Biotech Corporation Polymeric solid support for oligonucleotide synthesis

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US10138266B2 (en) 2015-06-18 2018-11-27 Nitto Denko Corporation Method of cutting out RNA oligonucleotide

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